Histoplasma capsulatum (Hc) is the dimorphic pathogenic fungus responsible for histoplasmosis, a pulmonary or systemic disease widespread throughout the Ohio-Mississippi River valley. Hc is an increasingly important opportunistic infection in immunodeficient patients, particularly the group with AIDS. Recently, Hc strains obtained from three AIDS patients from St. Louis area were classified with the low virulence temperature sensitive DOWNS strain. This finding, extended by the molecular and virulence analyses of additional patients isolates described in this study, may be yet another example of the dramatic susceptibility of these patients to disease caused by intercellular pathogens. Using these isolates and the dimorphism of Hc as tools, a model system will be used to isolate, identify and characterize virulence genes. The ability of Hc to exist in a mycelial (M) or yeast (Y) form will be exploited by the development of phase specific subtraction libraries to clone genes expressed early and late during the M-Y transition. Since the Y form is found exclusively in infected tissues and organisms blocked in the M-Y conversion are non-infectious in mice, these cloned genes may encode important Hc virulence determinants. Expression of one yeast phase specific gene, yps-3, correlates with virulence and temperature sensitivity in three non-isogenic Hc strains. Since the yps-3 gene is apparently Hc-specific, the feasibility of cloned sequences from this gene serving as diagnostic probes will be examined by polymerase chain reaction methodology. Virulence of Hc isolates obtained from patients with AIDS will be examined through comparisons of a previously developed in vivo murine model and an alternative in vitro assay, and will be correlated with the thermal susceptibilities of these organisms with reference strains. A transformation system for Hc will be developed by exploiting new technologies for protoplast formation and high efficiency electroporation, and by applying a benomyl resistance marker in a dominant selection procedure. Putative virulence genes identified from the subtraction libraries will be cotransfected with the benomyl marker into low virulence recipient Hc strains, such as DOWNS or one of the AIDS isolates Virulence of benomyl resistant transformants will be assayed in the in vivo and in vitro models. By integrating these technologies, this study should provide important insights into the virulence determinants operative in histoplasmosis among normal patients and in the nature of the defect in host resistance in individuals with AIDS.